PCR Amplification from clones - (Feb/06/2007 )
Hi all. I have Topo-cloned some bacterial PCR products. Now I want to amplify the insert with the help of SP6 and T7 promoter primers. The thing is that i see two equally spaced bands in all samples when i run the PCR products on agarose gel. While I am expecting only one band of about 500 bps. Can anyone help me please why there are two bands on the gel ????
non specific binding, perhaps?
hi blue, thanks for the post. we know it is non-specific binding but what can be the reason for it??? SP6 and T7 are vector specific primers and the vectors are supposed to have only our pcr amplified insert, which is about 700 bp. The second band, that i dnt want to see at all, is about a couple of hundred base pairs smaller. is it possible that E. coli rDNA is also there in the inserts ??? if yes how i can get rid of it ?
hi ... i was expecting some suggestion from you people .... anyway thanks for having atleast a look ....... uptil now i had 74 viewrs and 1 reply ... thats nice anyway but not nice enough
Have you done miniprep of your DNA to check if your plasmid is right? rDNA can be present but I would doubt you would see it on a gel after colony-PCR as you only add small amounts of bacteria to your PCR reaction.
firstly, have you confirmed that the plasmid and insert are correct by restriction digest?
Which vector are you using? pCRII? If so, what happens when you try a PCR with M13 primers?
Could one of the primers be binding within your insert sequence? Sounds to me like thats the most likely explanation is everything else is correct...
hi various and scrat... thanks for the posts. i tried M13 forward and reverse primers today ....... and got sharp single melting peaks in a RT-PCR reaction. ya most probably one of the primers was binding within the insert. M13s are good ..... i just need to order these primers ... thanks for ur help
yeah, M13 primers rock. They are your friends
Glad it worked!